Prevotella cerevisiae sp. nov., beer-spoilage obligate anaerobic bacteria isolated from brewery wastewater.

Two obligate anaerobic, Gram-stain-negative, non-spore-forming bacilli (strains SBC 8034T and SBC 8065) were isolated from brewery wastewater. Cells of the two strains were rod-shaped and 0.8×2-5 µm in size. Strains SBC 8034T and SBC 8065 did not grow on Columbia agar or tryptic soy agar II with 5 % sheep blood, brain-heart infusion agar or chocolate agar, but did grow on peptone-yeast-glucose agar and de Man, Rogosa and Sharpe agar using beer instead of water. The organisms produced acetic acid and succinic acid as the major metabolic end-products. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains are clearly distinct from all recognized species within the genus Prevotella, but belong to the same species (DDH=85 %). Based on 16S rRNA and hsp60 gene sequencing, along with phenotypic, chemical and biochemical properties, strains SBC 8034T and SBC 8065 were considered to represent a novel species within the genus Prevotella, for which the name Prevotellacerevisiae sp. nov. is proposed. Strain SBC 8034T (=DSM 100619T=JCM 30867T) is the type strain of the proposed novel species.

Analysis of mitochondrial function in human induced pluripotent stem cells from patients with mitochondrial diabetes due to the A3243G mutation.

We previously established human induced pluripotent stem (iPS) cells in two diabetic patients from different families with the mitochondrial A3243G mutation and isolated isogenic iPS cell clones with either undetectable or high levels of the mutation in both patients. In the present study, we analyzed the mitochondrial functions of two mutation-undetectable and two mutation-high clones in each patient through four methods to assess complex I activity, mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production. In the first patient, complex I activity, mitochondrial respiration, and mitochondrial ATP production were decreased in the mutation-high clones compared with the mutation-undetectable clones, and mitochondrial membrane potential was decreased in a mutation-high clone compared with a mutation-undetectable clone. In the second patient, complex I activity was decreased in one mutation-high clone compared with the other clones. The other parameters showed no differences in any clones. In addition, the complex I activity and mitochondrial respiration of the mutation-undetectable clones from both patients were located in the range of those of iPS cells from healthy subjects. The present study suggests that the mitochondrial function of the mutation-undetectable iPS cell clones obtained from two patients with the A3243G mutation is comparable to the control iPS cells.

Development of ghrelin transgenic mice for elucidation of clinical implication of ghrelin.

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of ß-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

An increase in the circulating concentration of monocyte chemoattractant protein-1 elicits systemic insulin resistance irrespective of adipose tissue inflammation in mice.

Chronic inflammation in adipose tissue is thought to be important for the development of insulin resistance in obesity. Furthermore, the level of monocyte chemoattractant protein-1 (MCP-1) is increased not only in adipose tissue but also in the circulation in association with obesity. However, it has remained unclear to what extent the increased circulating level of MCP-1 contributes to insulin resistance. We have now examined the relevance of circulating MCP-1 to the development of insulin resistance in mice. The plasma concentration of MCP-1 was increased chronically or acutely in mice to the level observed in obese animals by chronic subcutaneous infusion of recombinant MCP-1 with an osmotic pump or by acute intravenous infusion of MCP-1 with an infusion pump, respectively. Whole-body metabolic parameters as well as inflammatory changes in adipose tissue were examined. A chronic increase in the circulating level of MCP-1 induced insulin resistance, macrophage infiltration into adipose tissue, and an increase in hepatic triacylglycerol content. An acute increase in the circulating MCP-1 concentration also induced insulin resistance but not macrophage infiltration into adipose tissue. In addition, inhibition of signaling by MCP-1 and its receptor CCR2 by administration of a novel CCR2 antagonist ameliorated insulin resistance in mice fed a high-fat diet without affecting macrophage infiltration into adipose tissue. These data indicate that an increase in the concentration of MCP-1 in the circulation is sufficient to induce systemic insulin resistance irrespective of adipose tissue inflammation.

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte.

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

Association of serum MCP-1 concentration and MCP-1 polymorphism with insulin resistance in Japanese individuals with obese type 2 diabetes.

Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) secreted by adipocytes is a member of the CC chemokine family and plays a pivotal role in the inflammatory process. A polymorphism, the -2518 A/G of MCP-1 gene, has been associated with type 2 diabetes, type 1 diabetes, parameters of insulin resistance and obesity. Therefore, we investigated the effects of MCP-1 single nucleotide polymorphisms (SNPs) on the susceptibility to type 2 diabetes or insulin resistance in the Japanese population. We also assessed the correlation between serum MCP-1 concentration and other clinical characteristics in Japanese type 2 diabetic subjects. The serum MCP-1 concentration was significantly correlated with HOMA-IR and the visceral fat area, but not with BMI. Although there was no association between this SNP and type 2 diabetes, the -2518A/G polymorphism was associated with the serum MCP-1 concentration. In subgroup analysis, Japanese obese diabetic -2518AA carriers had a higher MCP-1 concentration and increased insulin resistance than obese diabetic -2518G carriers. These data indicated that the MCP-1 polymorphism was associated with insulin resistance in Japanese obese diabetic subjects and that MCP-1 was implicated in the pathogenesis of insulin resistance, especially associated with obesity, in humans.

Insulin-stimulated fusion of GLUT4 vesicles to plasma membrane is dependent on wortmannin-sensitive insulin signaling pathway in 3T3-L1 adipocytes.

It is established that wortmannin which completely inhibits class IA PI 3-kinase activation abrogated the insulin-dependent translocation of GLUT4 to the plasma membrane in adipocytes and skeletal muscle. However, it was not clear which steps wortmannin inhibited during the whole translocation process of GLUT4. We have now dissected the each steps of the GLUT4 trafficking in 3T3-L1 adipocytes using exogenously-expressed GLUT4 reporter in combination with plasma membrane lawn assay. We showed that 100 nM wortmannin inhibited the fusion of GLUT4 vesicles to the plasma membrane without affecting the movement and the subsequent tethering/docking event of GLUT4 vesicles to the membrane in 3T3-L1 adipocytes. These results suggest that wortmannin-sensitive insulin signaling pathway plays a crucial role in the fusion step of GLUT4 vesicles to the plasma membrane in 3T3-L1 adipocytes.

MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity.

Adipocytes secrete a variety of bioactive molecules that affect the insulin sensitivity of other tissues. We now show that the abundance of monocyte chemoattractant protein-1 (MCP-1) mRNA in adipose tissue and the plasma concentration of MCP-1 were increased both in genetically obese diabetic (db/db) mice and in WT mice with obesity induced by a high-fat diet. Mice engineered to express an MCP-1 transgene in adipose tissue under the control of the aP2 gene promoter exhibited insulin resistance, macrophage infiltration into adipose tissue, and increased hepatic triglyceride content. Furthermore, insulin resistance, hepatic steatosis, and macrophage accumulation in adipose tissue induced by a high-fat diet were reduced extensively in MCP-1 homozygous KO mice compared with WT animals. Finally, acute expression of a dominant-negative mutant of MCP-1 ameliorated insulin resistance in db/db mice and in WT mice fed a high-fat diet. These findings suggest that an increase in MCP-1 expression in adipose tissue contributes to the macrophage infiltration into this tissue, insulin resistance, and hepatic steatosis associated with obesity in mice.

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization.

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c-/- mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c-/- cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c-/- cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.